Project Summary Pulmonary hypertension (PH) is a multifactorial life threatening pulmonary vascular disease characterized by aberrant muscularization of the normally non-muscularized distal pulmonary arterioles (PAs). Dysfunction of PA endothelial cells (ECs) plays key roles in accumulation of pulmonary artery smooth muscle cells (PASMCs) to distal PAs. Expression of the transcription factor Twist1 is upregulated in the lungs of PH patients. However, the role of endothelial Twist1 in pathogenesis of PH has not been fully understood. The goal of this proposal is to explore the mechanism by which endothelial Twist1 controls accumulation of PASMCs to distal PAs. Our new preliminary data demonstrate that Twist1 overexpression increases the levels of platelet-derived growth factor B (PDGFB) in human pulmonary arterial endothelial (HPAE) cells and induces proliferation and migration of PASMCs. Hypoxia increases Twist1 Ser42 phosphorylation and PDGFB expression in HPAE cells and conditioned medium from these cells stimulates migration of PASMCs, while Twist1 knockdown in ECs inhibits the effects. When we implanted fibrin gel supplemented with fluorescently labeled ECs on the mouse lung, Twist1 overexpression in these ECs increases PDGFB expression and promotes the recruitment of ?-smooth muscle actin (SMA)-positive cells in the gel. Hypoxia induces accumulation of ?SMA-positive cells to blood vessels formed in the gel, while Twist1 knockdown in ECs attenuates the effects. Hypoxia also stimulates accumulation of ?SMA-positive cells to small PAs in the mouse lungs, which is suppressed in Tie2-specific Twist1 knockout mice. The levels of Twist1 and PDGFB are higher in lung ECs isolated from mice with type-2 bone morphogenetic protein receptor (Bmpr2) mutation that influences the severity of human PH. The Bmpr2 mutation also stimulates accumulation of ?SMA-positive cells to ECs in the gel implanted on the hypoxia- treated mouse lungs. We hypothesize that endothelial Twist1 mediates hypoxia-induced SMC accumulation to distal PAs through PDGFB paracrine signaling. In Aim 1, we will investigate whether endothelial Twist1 or its phosphorylation controls PASMC behaviors through PDGFB paracrine signaling in vitro. In Aim 2, we will determine whether endothelial Twist1 mediates hypoxia-induced SMC accumulation to blood vessels in the mouse lung using the mouse lung fibrin gel implantation system. We will also explore whether endothelial Twist1 mediates hypoxia-induced vascular remodeling using inducible VE-cadherin-specific Twist1 knockout mice. In Aim 3, we will investigate whether endothelial Twist1 mediates Bmpr2 mutations-induced vascular remodeling in ECs under normoxia or hypoxia in vitro and in the gel implantation model. Our focus on exploring the role of endothelial Twist1 in hypoxia-induced SMC accumulation to distal PAs using the mouse lung gel implantation system is unique and relevant advances, which will further our understanding of the pathogenesis of PH.